The overall hypothesis of this proposal is that tinman homologues are required for cardiac and visceral mesoderm specification, and for initiation and maintenance of the differentiated cardiac state, and that they fulfill these functions as either transcriptional activators or repressors in a manner dependent on their spatial and temporal developmental context. Although previous results by ourselves and others indicate that tinman homologues are important for cardiac development, many questions remain as to their in vivo roles. Our hypothesis is designed to address some of these questions. Accordingly, we will focus our studies on the action of two tinman homologues, XNkx2.5, and XNkx2.3, in developing Xenopus laevis embryos. Xenopus is an experimentally powerful system for studying early events of mesoderm specification and cardiac differentiation. Although expression of other tinman homologues has been described in developing heart, Nkx2.5 and Nkx2.3 are the best characterized, and appear to exhibit the most consistent expression in cardiac and visceral primordia and in adult heart across species. We will initially characterize and dissect transcriptional properties of XNkx2.5 and XNkx2.3 by transfection studies in mammalian tissue culture systems. Well-defined wildtype and mutant tinman constructs resulting from these studies will be used for ectopic expression studies in developing Xenopus embryos to analyze the role and mechanism of action of tinman homologues in both mesoderm specification and initiation and maintenance of the differentiated cardiac state. During the course of these studies, we have developed a novel strategy for transient trangenesis in Xenopus which allows for high levels of ectopic gene expression throughout the embryo, and in a tissue specific manner when directed by a tissue specific promoter. This strategy greatly facilitates ectopic expression studies in Xenopus, and may be of general applicability to ectopic expression studies in other systems, such as cell culture systems (including ES cells), and other developing embryos.